Mutations/variations in the human genome are involved in most diseases, going from monogenetic to multifactorial diseases, and acquired diseases such as cancer. Even the susceptibility to infectious diseases, and the response to pharmaceutical drugs, is affected by the composition of an individual's genome. Most genetic tests, which screen for such mutations/variations, require amplification of the DNA region under investigation. However, the size of the genomic DNA that can be amplified is rather limited. For example, the upper size limit of an amplified DNA fragment in a standard PCR reaction is about 2 kb. This contrasts sharply with the total size of 3 billion nucleotides of which the human genome is build up. As more and more mutations/variations are found to be involved in disease, there is a need for robust assays in which different DNA regions, that harbor the different mutations/variations, are analyzed together. This may be achieved through (more complex) multiplex amplification reactions.
In this application, PCR is the method of amplification of DNA that is mainly described, however the embodiment of this invention may be used for any amplification technique known to the art, such as isothermal amplification (rolling circle amplification), ligase chain reaction, nucleic acid sequence-based amplification (NASBA), padlock probes, single strand displacement amplification, and whole genome amplification.
In a classical multiplex PCR reaction, different fragments are amplified in a single tube, simply by adding all pairs of amplicon-specific primers to a reaction mixture. The higher the number of primers that are combined in a single PCR reaction, the higher the chance that particular primer interactions (such as primer-dimerization), and aspecific primer/template interactions occur, so that particular amplicons fail to amplify. There is thus a limitation in the number of amplicons that can be co-amplified when primers are simply mixed. Certain primer combinations work in one multiplex reaction, but not in another multiplex reaction. Also the amount of primers that are added may affect the success of a multiplex PCR amplification. The determination of amplicons that can be co-amplified, and the development of a robust multiplex PCR reaction of these amplicons, needs to be determined empirically by trial and error and is rather time-consuming. Each multiplex PCR reaction needs its own optimization. In a robust multiplex PCR reaction, 5-15 amplicons can be combined. A higher number of amplicons may be multiplexed, however at the expense of the robustness of the assay which affects the success rate of a multiplex amplification.
Robust multiplex PCR reactions of a large number of amplicons may be achieved when the different primers are physically restricted. Here we propose different methods to trap molecular components allowing a physically restricted amplification with minimal interference of other molecular components.
New sequencing techniques allow a very large throughput and the generation of large amounts of data. They may even generate much more sequence data than needed for the analysis of one sample. In order to make full use of the capacity of a technique, and therefore at an economical cost, different samples need to be pooled in one single sequencing run. This requires that the origin of the DNA fragments derived from the respective samples can be traced. Addition of a DNA-tag to a DNA fragment of a sample that one wants to analyze can be obtained in a one step amplification process in which at least one amplicon-specific primer contains the tag as an adaptor. However, this will be still very costly since the number of primers will increase very rapidly with increasing number of samples and increasing number of amplicons to be analyzed per sample.
Prior Art: